Broad Range Polymerase Chain Reaction for Diagnosis of Rat-Bite Fever Caused by Streptobacillus Moniliformis

An 11-year-old boy presented with fever, vomiting, rash, limping and blisters on his feet after a finger bite by his domestic rat. Although cultures from blood, cerebrospinal fluid and urine remained negative, broad range polymerase chain reaction amplification of a part of the 16S rRNA gene followed by sequencing allowed the detection and identification of Streptobacillus moniliformis in blister fluid, thus confirming the suspected clinical diagnosis of rat-bite fever. Rat-bite fever is a systemic illness caused by infection with Streptobacillus moniliformis or Spirillum minus that is acquired through the bite or scratch of a rodent or by ingestion of contaminated food. Streptobacillary rat-bite fever is characterized by relapsing fever, asymmetric polyarthritis and maculopapular rash on the extremities. Etiologic diagnosis is established by blood culture. However, this is difficult and sometimes unsuccessful due to the fastidious growth of the organism and the need for special growth media. We report on a boy with rat-bite fever caused by S. moniliformis as detected in the blister fluid by broad range PCR. Case report. An 11-year-old Swiss boy was admitted to our emergency room because of a 4-day history of relapsing fever up to 39.5°C, headache, vomiting, transient erythematous rash on arms and legs and limping. He had been seen 3 days before in a regional hospital where he complained of migratory arthralgias in the right wrist, knee and both ankles. A maculopapular rash on his extremities and a normal cerebrospinal fluid (CSF) examination were noted. A viral infection was suspected, and the boy was discharged 2 days later without antibiotic treatment. Fever relapsed, arthralgias persisted and several serous blisters emerged on the back and medial ankle of his feet. He contacted the regional hospital that referred him to our emergency unit. He recalled a tick bite 3 weeks earlier without subsequent erythema and a bite in his finger by the recently acquired pet rat 7 days before. Physical examination revealed an ill-appearing boy with an axillary temperature of 38.4°C, a modestly stiff neck, erythema on his arms and legs and painful tender swelling of both ankles, the right elbow, wrist and metacarpophalangeal joints. On both sides of the left ankle serous blisters, the largest 0.7 cm in diameter and hemorrhagic, were noted. The bite wound puncture on his right ring finger was dry and crusted. The leukocyte count was 5.6 × 10 9/l with 61.5% neutrophils, 20% lymphocytes and 18% monocytes; hemoglobin 114 g/l; platelets 147 × 10 9/l; the erythrocyte sedimentation rate 39 mm/h; and serum C-reactive protein 87 mg/l. Liver enzymes and creatinine were normal. The CSF examination yielded 5 mononuclear cells/μl, normal glucose and protein concentrations and no microorganisms on direct microscopy after Gram staining. Blood, CSF, urine and the serous blister fluid were obtained for bacterial cultures. Rat-bite fever or leptospirosis was suspected. Intravenous penicillin (50 000 units/kg/day) was started. Alleviation of symptoms and defervescence followed rapidly. He started moving his joints after 2 days, but 1 day later his left hip ached. Ultrasound examination revealed an effusion. After 7 days treatment was switched to oral penicillin, and 3 days later the afebrile and freely walking boy was discharged with dry skin lesions. Penicillin was discontinued after a total treatment of 17 days. When seen 4 weeks after admission, the boy was well, all lesions had healed and C-reactive protein and erythrocyte sedimentation rate were normal. Microbiology. Aerobic and anaerobic blood cultures were incubated at 37°C in the BACTEC 9240 system (Becton Dickinson Microbiology Systems, Basel, Switzerland) for 7 days and remained sterile, as did cultures from CSF, blister fluid and urine. No serum antibodies to Borrelia burgdorferi and Leptospira were detected. Serologic tests for rat-bite fever were not available. We used a broad range PCR technique based on the amplification of bacterial 16S ribosomal RNA (rRNA), essentially as described previously, 1 to detect S. moniliformis in the blister fluid. A part of the 16S rRNA gene (corresponding to positions 8 to 806 of the Escherichia coli 16S rRNA gene) was amplified with broad range bacterial primers. Amplicons were reamplified with broad spectrum primers (covering positions 8 to 533) and sequenced using the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Rotkreuz, Switzerland). Partial sequences were compared with bacterial 16S rRNA sequences deposited in the GenBank and EMBL databases and revealed a sequence of 203 base pairs identical with that of S. moniliformis, whereas the most closely related organism , Leptotrichia sanguinensis, was only 94% related. Other organisms, including typical skin pathogens and colonizers, exhibited homology below 90% to the sequence determined. Discussion. Infections after rat bites include leptospirosis and rat-bite fever. The latter is a systemic bacterial illness caused by S. moniliformis or by Spirillum minus.2, 3 The clinical syndrome develops after 2 to 10 days of incubation with relapsing fever, asymmetric polyarthritis and an ensuing rash on the extremities. Endocarditis, meningitis or pneumonia and focal abscesses may follow. 2–4 Untreated cases are fatal in 13%. 3 The diagnosis of streptobacillary rat-bite fever is classically established by blood culture. However, this is difficult in routine practice, because of the fastidious nature and the slow growth of these Gram-negative facultative anaerobic rods and the strict growth requirements including special media that are not readily available. 3–5 Serologic tests are unreliable and therefore are no longer recommended or available. 2 Identification of S. moniliformis in focal lesions, e.g. blisters, allows the diagnosis in blood culture-negative cases. This is particularly important in cases in which antimicrobial treatment has already been started. We believe that this is the first reported case demonstrating that broad spectrum PCR technique offers a novel additional means to detect the causative agent of rat-bite fever. The assessment of additional clinical specimens will prove the utility of this technique in clinical practice.